Efflux Pumps Contribute to Intrinsic Clarithromycin Resistance in Clinical, Mycobacterium abscessus Isolates

Figures & data

Table 1 MIC of Clarithromycin for 194 Clinical M. abscessus Isolates

M. abscessus	No. of Isolates	Number of Isolates Exhibiting the MIC (mg/L) of CLA Indicated													No. of Resistant Isolates (%)	Resistant Isolates Harboring a rrl
		0.06	0.125	0.25	0.5	1	2	4	8	16	32	64	128	256		2270/2271 Mutation^a
subsp. abscessus	148	16	17	10	30	34	27	6	2	1	1	2	2	0	8 (5.4)	4
subsp. massiliense	46	23	10	3	2	2	1	0	0	0	0	2	1	2	5 (10.9)	3

Note: ^aE. coli numbering 2058 and 2059.

Table 2 Blast Search of the M. abscessus Genome with Reference Gene Sequences Corresponding to Efflux Pumps Conferring or Putatively Involved in CLA Resistance

Targeted Gene	Sequence in Reference Strain (NC_010397.1)^a	Gene Description	Reference Gene		Reference(s)
			Homolog	Strain
MAB_2355c	2410335-1993	Putative ABC transporter ATP-binding protein	Rv1473 MSMEG_3140 MAV_3306	M. tuberculosis M. smegmatis M. avium	^Citation15,Citation16
MAB_1409c	1412431-3702	Putative drug antiporter protein precursor	Rv1258c (tap) MAV_1406	M. tuberculosis M. avium	^Citation17,Citation18
MAB_1846	1844817-6403	Putative ABC transporter ATP-binding protein	Rv2477c	M. tuberculosis	^Citation18,Citation19
MAB_3142c	3180588-2105	Putative transmembrane efflux protein	Rv2846c(efpA)	M. tuberculosis	^Citation20,Citation21
MAB_2807	2854903-6447	Major facilitator superfamily MFS_1	Rv1410c (p55)	M. tuberculosis	^Citation22,Citation23
MAB_0970c	976402-8414	Probable drug resistance transporter	Rv1877	M. tuberculosis	^Citation20,Citation21
MAB_1560	1587054-8727	Probable ABC transporter (macrolide-transport) ATP-binding protein	MAV_1695	M. avium	^Citation16

Note: ^aComplete nucleotide sequence in the M. abscessus genome NCBI database.

Figure 1 Efflux pump gene expression by clinical, M. abscessus isolates following exposure to CLA stress. Isolates that were CLA-susceptible, -intermediate and resistant [without (unexplained) or with a rrl 2270/2271 mutation] are shown. MAB_2355c (A), MAB_1409c (B), MAB_1846 (C) and whiB7 (D), message levels were quantified by qRT-PCR. Open circles, triangles, diamonds, and solid circles indicate the distribution of mRNA expression levels in isolates comprising the sensitive, intermediate, unexplained drug resistance, and rrl 2270/2271 mutation groups, respectively. Experiments were repeated >3 times for each isolate; error bars represent the standard error of the mean for each data set.

Table 3 Efflux Pump Inhibition Decreases the MIC of Clarithromycin for Non-Susceptible Isolates^a

Treatment	Number (%) of Isolates Exhibiting a Significant Decrease in CLA Resistance^b
	Intermediate (n = 6)	Resistant with rrl Mutation (n =7)	Resistant Without rrl Mutation (n = 6)	Total (n=19)
CLA + PaβN	4 (66.7)	0 (0)	5 (83.3)	9 (47.4)
CLA + CCCP	5 (83.3)	0 (0)	5 (83.3)	10 (52.6)
CLA + VP	5 (83.3)	1 (14.2)	6 (100)	12 (63.2)

Notes: ^aIsolates exhibiting MIC ≥4 mg/L after 3 days incubation were considered non-susceptible. ^bAfter the addition of the inhibitor, a decrease in MIC of ≥2-fold was considered significant.

Abbreviations: PaβN, phenylalanine-arginine β-naphthylamide; CCCP, Carbonyl cyanide 3-chlorophenylhydrazone; VP, verapamil.

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